PROJECT SUMMARY The Nobel winning discoveries of Toll-like Receptors (TLRs) and interleukin-1 receptors (IL-1Rs) have revolutionized our understanding of inflammation and oncogenesis, which makes it surprising that the initiating intracellular events still remain poorly characterized. Specifically, both receptor families share common intracellular Toll/Interleukin-Receptor (TIR) domains that engage adaptor TIR domains in order to initiate signaling, yet no human oligomeric TIR complex has been structurally observed. Instead, the TIR interactome has almost exclusively been extrapolated from structures of individual TIR domains and computational modeling, often resulting in conflicting data. Thus, despite the nearly two decades since the first structural characterization of the TIR domains of TLR1 and TLR2 receptors, the molecular mechanisms that underlie the critical roles of TIR signaling remains unknown. Our goals are to determine the molecular basis of TIR domain interactions that underlie the innate immune response, thereby bridging the initiating events on the outside of the cell with downstream events that drive inflammation. The novelty in our approach is our combination of biochemical, biophysical, and biological studies along with our unique ability to recombinantly produce multiple human TIR members, which has revealed a surprising underlying molecular mechanism of TLR interactions. Namely, we have discovered that TLR1/TLR2 homodimer and heterodimer formation are mediated by an intermolecular disulfide exchange of a conserved cysteine found in all TLRs expressed on the cellular surface. Such a signaling mechanism, referred to as ?dock-and-lock?, has only been observed for PDZ domains that also form signaling complexes at the cellular membrane. However, disulfide mediated TIR interactions have been observed in other organisms and a drug specific for the same conserved cysteine within TLR4 blocks its activity. Such studies highlight the importance of determining the molecular mechanism of TLR interactions that would also provide a basis for pharmacologically blocking their interactions through targeting of this conserved cysteine. Interestingly, our preliminary studies also suggest that the anti-inflammatory activity of the orphaned IL-1R8 receptor blocks TLR1/TLR2 interactions through a similar ?dock-and-lock? mechanism, providing the molecular basis for one of the most exciting negative regulators of inflammation within the last 15 years. Based on these preliminary studies, we hypothesize that a conserved cysteine mediates complex formation of TLR1, TLR2, and IL-1R8 through a ?dock-and-lock? mechanism. The versatility in homo/heterodimerization results in varied downstream TIR interactions that fine-tune the cellular inflammatory response. We will address this hypothesis through the following Specific Aims: Aim 1) Determine the molecular basis of TLR1/TLR2 TIR homodimerization and heterodimerization and how their specific interactions regulate cellular signaling. Aim 2) Determine how IL-1R8 TIR blocks TLR1/TLR2 TIR interactions and how such interactions modulate downstream adaptor interactions.